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Transcriptional Regulation
We are interested in how gene expression is controlled in eukaryotes. Our main research goal is to understand molecular mechanisms responsible for assembly and disassembly of transcription preinitiation complexes at promoters, a major regulatory step in the complex process of RNA synthesis. Most of our work exploits the budding yeast system, although we also work with human cells as well.

Chromatin binding dynamics
Our analysis of transcription preinitiation complex assembly led us to consider fundamental questions about chromatin binding dynamics in cells. We seek to understand not only pathways for assembly and activity of transcription complexes in vivo, but the time scales on which these processes occur, how efficient they are in a cell population, and how stable complexes are once they assemble on chromatin. To address these questions, we developed a method that allows us to measure chromatin binding kinetics at specific chromosomal sites in cells.

Research

Transcriptional regulatory mechanisms involve a complex interplay of factors whose effects are mediated by physical interactions among them and diverse enzymatic activtites, all occurring in the context of the native chromatin fiber. To understand aspects of this regulation we apply approaches that span the full range from biochemistry, biophysics and structural biology, to molecular biology, cell biology, genetics and genomics.

For investigation of transcriptional regulatory mechanisms in cells, we use genetic and molecular biological approaches to engineer strains with particular defects, and we characterize the effects of these mutations on transcription by measuring transcription factor interactions with chromatin and RNA output at particular genes as well as genome-wide.

An essential and conserved regulator of transcription preinitiation complex (PIC) assembly and activity is an enzyme called Mot1. It interacts with the TATA-binding protein (TBP), a central component of the PIC, and has the remarkable property of using ATP hydrolysis to remove TBP from DNA. Mot1 is comprised of two functional domains. Its N-terminal domain (NTD) has an extended corkscrew-like shape that interacts with the saddle-shaped TBP molecule. The image on the home page shows the co-crystal structure of the NTD-TBP complex. The C-terminal region of Mot1 possesses an ATPase domain that is highly related to a large number of ATPases belonging to the Snf2/Swi2 family. These enzymes play critical roles in all aspects of DNA metabolism including transcription, replication and repair. In general, enzymes in this family use ATP hydrolysis to catalyze changes in protein-DNA interactions, and understanding how they do this on a molecular level has been the subject of intense interest by many labs. We apply biochemical, biophysical and structural biological approaches to better understand the Mot1 catalytic mechanism, and in so doing seek to better understand how enzymes in this important class function in general.

Our recent work on Mot1 and PIC dynamics showed that in general, the interactions between general transcription factors and chromatin are surprisingly short-lived in vivo. Biochemical studies, however, had established that a subset of PIC components can be stably bound to promoters in vitro, nicely explaining in principle how the activated state of a gene can be perpetuated. We wondered what the relationship of the rapid dynamics we observed could be to the normal process of gene expression and regulation. One possibility was that the biochemical studies did not accurately capture the dynamic processes occuring in vivo. Another possibility was that binding dynamics vary, potentially dramatically, at different promoters in vivo- an idea with interesting implications for gene regulation. Alternatively, stable complexes may form in vivo, but perhaps they do so inefficiently. In support of a fundamentally important role for chromatin binding dynamics, we found that Mot1-mediated TBP turnover is essential genome-wide for accurate and efficient transcription, and perturbing it can impact apparently all steps of the process of RNA synthesis- from initiation, to elongation, to termination. For this reason, a major focus of our current work is to develop and exploit methods for measuring chromatin binding dynamics at single copy genes in vivo. In a recently developed method which we call Crosslinking Kinetics (or CLK, pronounced "clock") Analysis we measure how the chromatin immunoprecipitation (ChIP) signal depends on the formaldehyde incubation time. By modeling this process we can obtain estimates of the on-rate, the off-rate and the fractional occupancy for chromatin binding, among other parameters. So far, we have seen that chromatin complexes span an enormous range of stability, with half-lives ranging from just a few seconds to an hour or more. How regulators influence this dynamic behavior is just beginning to be explored, but we have already observed that quantitative analyses of chromatin binding in vivo by our methods can challenge models derived from conventional ChIP studies and provide new insight into regulatory mechanisms operating at promoters.